Abstract

<Background> Clinically, 5-fluorouracil (5-FU) based chemotherapy has been proven to be effective for colorectal cancer patients in either adjuvant or metastatic setting. In the adjuvant setting, it remains unresolved issues whether 5-FU inhibits the adhesion of circulating tumor cells to the target organs such as liver or lung, or it eradicates clinically undetectable micrometastases in these organs. <Aim> We examine whether 5-FU inhibits the adhesion of tumor cells to the liver on murine colorectal liver metastasis model using intravital multiphoton microscopy. We also examine whether 5-FU inhibits the liver metastasis formation from a single tumor cell to metastatic colonies in the same mice using time-series intravital multiphoton microscopy. <Materials and methods> Red fluorescent protein (RFP) expressing SL4 murine colorectal cancer cell line (RFP-SL4) and green fluorescent protein (GFP)-transgenic mouse (GFP mouse) were used. 3×106 RFP-SL4 cells were injected into the spleen of GFP mice for syngeneic liver metastasis model. Intravital multiphoton microscopy was performed by exteriorizing the liver lobe of the living mice and fixing it with a solder lug terminal and an adhesive agent. Time-series imaging of the living liver of the same mice was performed using intravital multiphoton microscopy at multiple time points. 5-FU was administered before or after the inoculation of RFP-SL4 cells to examine its inhibitory effect for tumor cell adhesion or liver metastasis formation, respectively. <Results> The arrested tumor cells were clearly observed in hepatic sinusoids 2 hours after inoculation. Time-series imaging of the same mice showed that the number of tumor cells decreased in time dependent manner (2h, 24h, and 72h after inoculation). Approximately 20 tumor cells were imaged 2 hours after inoculation in the voxel volume of 210×210×100 μm (x, y, z). There is no significant difference in the number of imaged tumor cells between the 5-FU pre-treatment and the control group by tumor volumetric analysis (2h after inoculation). All mice (100%) had liver metastases with diffuse growth pattern at 2 weeks after inoculation in the control group. After observing the adhesion of tumor cells to hepatic sinusoids by 1st intravital microscopy, the only half (50%) of mice had liver metastases in the 5-FU treatment group macroscopically and microscopically (2nd intravital microscopy at 2 weeks after inoculation). <Conclusions> 5-FU based adjuvant chemotherapy may suppress colorectal liver metastasis by inhibiting the process from a single tumor cell to metastatic colonies. Time-series intravital multiphoton microscopy enables us to visualize the treatment efficacy at the cellular level in the same animals with the reduction of inter-individual variation.